Gene encoding endochitinase

ABSTRACT

Two chitinases from Trichoderma harzianum P1 (ATCC 74058) show chitin-containing-fungus-inhibiting activity. One is an endochitinase and the other is a chitobiase. Both have molecular weights of 40 kDa and isoelectric points of 3.9. Endochitinases and chitobiases including the two purified from Trichoderma harzianum strain P1 demonstrate synergy with each other in antifungal effect. Isolated gene encoding for the endochitinase has the sequence set forth in the Sequence Listing as SEQ ID NO:1.

This invention was made in part with Government support under U.S. -Israel International Agricultural Research and Development Fund (BAPD)grant number US-1723-89. The Government has certain rights in theinvention.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of U.S. Ser. No. 08/045,269, filed Apr.14, 1993, now U.S. Pat. No. 5,378,821, which is a continuation-in-partof U.S. Ser. No. 07/919,784 filed Jul. 27, 1992, which is acontinuation-in-part of U.S. Ser. No. 07/716,134, filed Jun. 17, 1991,now U.S. Pat. No. 5,173,419

And a continuation-in-part of U.S. Ser. No. 08/184,115, filed Jan. 21,1994, now abandoned, which is a continuation-in-part of Ser. No.08/049,390, filed Apr. 21, 1993, now U.S. Pat. No. 5,474,926, which is acontinuation-in-part of U.S. Ser. No. 07/990,609, filed Dec. 15, 1992,now U.S. Pat. No. 5,326,561.

TECHNICAL FIELD

This invention is directed to isolation of chitinases for biologicalcontrol of chitin-containing fungi and insects.

BACKGROUND OF THE INVENTION

Application of broad-spectrum pesticides is the primary method used forcontrolling fungal and insect pests. Such application has resulted insignificant environmental pollution and ecological disruption. Pesticideresidues are found in food and groundwater and often eliminatebeneficial organisms resulting in emergence of secondary pests.Furthermore, as the target pests become less susceptible to thepesticide, there can be a resurgence of the original pest, requiringapplication of excessive quantities of pesticides for control.

A number of strategies for biological or biorational control of fungaland insect pests have been envisioned. Among the more attractivestratagies are those that target an attribute that is pest specific. Onetarget that has been selected is the structural polymer chitin, which ispresent in insects and some fungi that attack plants, but is absent inhigher plants and vertebrates. U.S. Pat. No. 4,751,081 follows thisapproach and is directed to novel chitinase-producing bacteria strainsfor use for inhibiting chitinase-sensitive plant pathogens (fungi andnematodes). The approach of U.S. Pat. No. 4,751,081 lacks flexibility.

SUMMARY OF THE INVENTION

An object of the invention herein is to provide purified chitinaseswhich can be used per se to inhibit fungi and insects that containchitin or can be used to provide novel chitinase-producing bacteria asin U.S. Pat. No. 4,751,081 or can be used to isolate genes coding forthem which can be inserted into a genome of a plant needing protectionfrom a chitin-containing pest.

The chitinases that are the subject of the instant invention areisolated from Trichoderma harzianum strain P1 . This strain wasdeposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852 on May 20, 1991, under the terms of theBudapest Treaty, and has been assigned accession number ATCC 74058.

The chitinases herein inhibit chitin-containing fungi and insects.

One chitinase herein is an essentially pure protein and hasendochitinase activity and has a molecular weight of 36 kDa (asdetermined by sodium dodecyl sulfate polyacrylamide gel electrophoresisafter the protein was prepared under reducing conditions, on directcomparison to migration of a 36 kDa protein) and an isoelectric point of5.3±0.2 as determined based on its elution profile from achromatofocusing column. It has a molecular weight of 40 kDa (asdetermined by sodium dodecyl sulfate polyacrylamide gel electrophoresisafter the protein was prepared under reducing conditions, from aregression based on the log of the molecular weight of standardproteins) and an isoelectric point of 3.9 as determined by isoelectricfocusing electrophoresis from a regression of distance versusisoelectric point of standard proteins. It has an optimum activity atabout pH 4 with a gradual decline to about pH 7. This chitinase issometimes described hereinafter as the endochitinase herein.

Another chitinase herein has exochitinase activity and has a molecularweight of 36 kDa (as determined by sodium dodecyl sulfate polyacrylamidegel electrophoresis after the protein was prepared under reducingconditions, on direct comparison to migration of 36 kDa protein) and anisoelectric point of 4.4±0.2 as determined based on its elution profilefrom a chromatofocusing column. It has a molecular weight of 40 kDa (asdetermined by sodium dodecyl sulfate polyacrylamide gel electrophoresisafter the protein was prepared under reducing conditions, from aregression based on the log of the molecular weight of standardproteins) and an isoelectric point of 3.9 as determined by isoelectricfocusing electrophoresis from a regression of distance versusisoelectric point of standard proteins. It has chitobiase activity, asindicated by its substrate specificity. It has an optimum activitybetween pH 4 and pH 7. This chitinase is purified to greater than a75-fold increase in specific activity compared to its activity in aculture filtrate of Trichoderma harzianum strain P1 having accession No.ATCC 74058. This chitinase may be obtained as an essentially pureprotein or in purified condition may be present with a minor amount,e.g., up to 40% by weight (total chitobiase basis), of a chitobiasehaving a molecular weight of 36 kDa (as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis after the protein wasprepared under reducing condition, from regression based on the log ofthe molecular weight of standard proteins) and an isoelectric point of3.9 as determined by isoelectric focusing electrophoresis from aregression of distance versus isoelectric point of standard proteins.This chitinase in the form of an essentially pure protein may bedescribed hereinafter as the chitobiase herein.

These chitinases are sometimes referred to collectively hereinafter asthe "purified chitinases herein" or as chitinases "of the inventionherein".

Where the molecular weight was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis after the protein was prepared underreducing condition, from regression based on the log of the molecularweight of standard proteins, the proteins were seven standard proteinsobtained from Sigma Chemical Co., having molecular weights ranging from14.2 to 66 kDa, and molecular weights were estimated from a regressionequation of the log of molecular weight of the standard proteins versusdistance migrated; the seven standard proteins and their molecularweights in kDa are respectively α-lactalbumin, 14.2; soybean trypsininhibitor, 20.1; trypsinogen, phenylmethylsulfonyl fluoride treated, 24;carbonic anhydrase, 29; glyceraldehyde-3-phosphate, 36; egg albumin, 45;and bovine albumin, 66. When the isoelectric point was determined byisoelectric focusing electrophoresis from a regression of distanceversus isoelectric point of standard proteins, comparison was to 12standard proteins obtained from Pharmacia LKB Biotechnology havingisoelectric points ranging from pH 3.5 to pH 9.3; the standard proteinsand their isoelectric points are respectively amyloglucosidase, 3.5;methyl red dye, 3.75; soybean trypsin inhibitor, 4.55; β-lactoglobulin,5.2; bovine carbonic anhydrase B, 5.85; human carbonic anhydrase B,6.55; horse myoglobin cynocytic band, 6.85; horse myoglobin basic band,7.35; lentil lectin acidic band, 8.15; lentil lectin middle band, 8.45;lentil lectin basic band, 8.65; and trypsinogen, 9.3. In both cases alinear regression was employed and r² values ranged from 0.94 to 0.99.For determination of pH optima, 50 mM citric acid and 50 mM of either K₂HPO₄ (endochitinase) or K₃ P0₄ (chitobiase) were prepared and these twosolutions were mixed in various ratios to give various pH values andassays were run in triplicate for each pH value andnitrophenyl-β-D-N,N'-diacetylchitobioside andnitrophenyl-β-D-N,N',N"-triacetylchitotriose were used as substrates forchitobiase and endochitinase respectively.

A further embodiment of the invention herein involves a biologicallypure (i.e., free of contaminating protein) composition containingendochitinase (enzyme that cleaves chitin randomly) and chitobiase(enzyme that cleaves dimeric units from chitin), preferably theendochitinase herein and the chitobiase herein, in a weight ratioranging from 3:1 to 1:1.2.

The term "essentially pure" is used herein to mean the presence of asingle protein band on a sodium dodecyl sulfate polyacrylamide gelsubmitted to electrophoresis under reducing conditions and stained withsilver stain. The term "in purified condition" means "essentially pure"with exception as stated.

The term "inhibit" is used herein to mean reduce the growth and/ordevelopment of fungi or insects compared to where inhibiting agent isnot present.

The term "endochitinase activity" is used herein to mean ability tocleave chitin randomly. Such activity is readily determined by an assayto measure reduction of turbidity of a suspension of purified chitin byan agent wherein reduction of turbidity indicates endochitinase activityfor the agent. The method of purification of the chitin is described inVessey, J. C., et al, Transact. Brit. Mycol. Soc. 60, 133-143 (1973) andinvolves grinding and washing crab shell chitin (Sigma Chemical Co.)with distilled water, washing with a mixture containing ethanol:diethylether:HCl (50:50:1), bleaching with NaOCl, dissolving in HCl,precipitating by diluting with ice water, and then repeatedly washingwith water adjusted to pH 8.5 until the pH of the chitin equals at least3. The assay involves the following: One g. of purified chitin issuspended in 100 ml 50 mM KHPO₄ buffer pH 6.7. To a test tube is added0.5 ml of this suspension; then 0.5 ml of the test sample is added. Thetube is incubated at 30° C. for 24 hours and then diluted with 5 mlwater. The optical density of a suspension is determined at 510 nm. Thepercentage reduction in turbidity is calculated relative to addition ofa sample without enzyme.

As used herein, the term "endochitinase" refers to enzymes that randomlycleave chitin.

The term "exochitinase activity" is used herein to mean ability tocleave chitin from one end. Such activity is readily determined bystandard assays by release of chromogenic p-nitrophenol fromp-nitrophenyl-N-acetyl-β-D-glucosaminide orp-nitrophenyl-β-D-N,N'-diacetylchitobiose, which substrates respectivelymeasure for activity of β-N-acetylglucosaminidase (nagase activity) andN,N'-diacetylchitobiase (chitobiase activity). The assays are the sameexcept for the substrate and involve the following: A substrate solutionis formed by dissolving 3 mg of substrate in 10 ml 50 mM KHPO₄ buffer pH6.7. Fifty μl of a substrate solution is added to a well in a microtiterplate (Corning). Thirty μl of test solution is added, and incubation iscarried out at 50° C. for 15 minutes. Then the reaction is stopped byaddition of 50 μl of 0.4 M Na₂ CO₃, and the optical density is read at410 nm. Enzyme solutions may have to be diluted, since optical densityreadings should be below 1.0. Activity is calculated as the opticaldensity×the dilution factor.

As used herein, the term "chitobiase" means enzyme that cleaves dimericunit from chitin. Chitobiase is sometimes described herein as "biase."As used herein the term "nagase" means enzyme that cleaves monomericunits from chitin. Both are identified using the enzyme assays describedabove.

A further aspect of the invention herein involves inhibiting thegermination of a chitin-containing fungus which comprises contactingsuch fungus with an antifungal effective amount of chitinase of theinvention herein or of the biologically pure composition hereincontaining endochitinase and chitobiase. In a preferred use, the fungusis from the genera Fusarium, Botrytis, Trichoderma (different fromTrichoderma harzianum stain P1), Uncinula or Ustilago.

A further aspect of the invention herein involves the λgtll recombinantcontaining a cDNA encoding for the endochitinase herein. This λgtllrecombinant was deposited with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852 on Jul. 6, 1992, under theterms of the Budapest Treaty as Bacteriophage P1, and has been assignedaccession number ATCC 55338.

A further aspect of the invention herein involves the gene encoding forthe endochitinase herein removed from the DNA of said λgtll recombinantby the restriction enzyme, Not I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the elution pattern for the concentrated broth of ExampleI and provides a graph of endochitinase activity (Fraction vs. %Reduction in Turbidity) denoted by x's, a graph of chitobiase activity(Fraction vs. Nitrophenyl activity) denoted by open boxes, and a graphof nagase activity (Fraction vs. Nitrophenyl activity) denoted byfilled-in diamonds.

FIG. 2 depicts the elution pattern for concentrated fractions of Set IIIof Example I and provides a graph of endochitinase activity (Fractionvs. % Reduction in Turbidity) denoted by filled-in diamonds, and a graphof Fraction vs. pH denoted by open boxes.

FIG. 3 depicts the elution pattern for concentrated fractions of Set IIof Example I and provides a graph of nagase activity (Fraction vs.Nitrophenyl activity) denoted by filled-in diamonds, a graph ofchitobiase activity (Fraction vs. Nitrophenyl activity) denoted by openboxes and a peak at fraction 20 and a graph of Fraction vs. pH denotedby x's and having a progressively descending path.

FIG. 4 depicts the elution pattern for the concentrated broth of ExampleIV and provides a graph of endochitinase activity (Fraction vs. % RedTurbidity, i.e., % Reduction in Turbidity) denoted by x's, a graph ofchitobiase activity (Fraction vs. Nitrophenyl activity) denoted byfilled in diamonds and a graph of nagase activity (Fraction vs.Nitrophenyl activity) denoted by open squares.

FIG. 5 depicts the elution pattern for the fractions of Set III ofExample IV and provides a graph of endochitinase activity (Fraction vs.% Reduction in Turbidity) denoted by open squares, and a graph ofFraction vs. pH denoted by x's.

FIG. 6 depicts the elution pattern for the fractions of Set II ofExample IV and provides a graph of nagase activity (Fraction vs.Nitrophenyl activity) denoted by open squares, a graph of chitobiaseactivity (Fraction vs. Nitrophenyl activity) denoted by filled indiamonds, and a graph of Fraction vs. pH denoted by x's.

FIG. 7 depicts the elution pattern for selected fractions fromchromatofocusing separation of Set II, separated according toisoelectric point on a Rotofor apparatus and provides a graph ofchitobiase activity (Fraction vs. Nitrophenyl activity) denoted byfilled-in diamonds, a graph of nagase activity (Fraction vs. Nitrophenylactivity) denoted by open squares and a graph of Fraction vs. pH denotedby x's. The graph shown is based upon 1.5 μl of enzyme rather than theusual 30 μl since the enzyme activity was high.

FIG. 8 depicts the synergy obtained with various combinations ofendochitinase and chitobiase and shows results of Example VII.

DETAILED DESCRIPTION

Trichoderma harzianum strain P1 (ATCC 74058) arose as a spontaneouslyoccurring isolate on placement of Trichoderma harzianum strain 107 on amedium containing 500 ppm of the fungicide iprodione by inventor A.Tronsmo. Strain 107 was isolated from wood shavings by Dr. C. Dennis inNorfolk, England and was selected by Tronsmo and Dennis as a coldtolerant isolate in a survey for biocontrol agents effective in coldclimates. Trichoderma harzianum strain P1 (ATCC 74058) has beenevaluated as a biocontrol agent as described in Tronsmo, A., NorwegianJournal of Agricultural Sciences, 3, 157-161, 1989 (biological controlof storage rot on carrots) and has been successfully used as abiocontrol agent of Botrytis cinerea, a fungus affecting strawberries,grapes and apples as described in Tronsmo, A., Biological Control, 1,59-62, 1991 and Harman, G. and Tronsmo, A., unpublished.

The purified protein chitinases herein are obtained from Trichodermaharzianum strain P1 (ATCC 74058) as follows: The strain is readilycultured, for example, in modified Richard's medium (composition in oneliter of water of 10 g KNO₃, 5 g KH₂ PO₄, 13 g MgSO₄, 20 mg FeCl₃, 10 gcrab shell chitin (Sigma Chemicals), 10 g Polyclar AT (an insolublepolyvinylpyrrolidone from GAF Corp) and 150 ml V8 juice (Campbell SoupCompany)) at 25° C. After 3 to 5 days of culturing, the hyphal mass isremoved to provide a broth which is dialyzed to remove small molecularweight molecules and then concentrated about 30-fold. The concentratedbroth is subjected to liquid chromatography to collect a fraction withchitobiase activity with low nagase activity and a fraction with onlyendochitinase activity. The fractions are concentrated and the proteinsare eluted using a chromatofocusing column. The turbidity reducingportion of the endochitinase activity fraction elutes as a single peakas the essentially pure protein having endochitinase activity, amolecular weight of 36 kDa (as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis after the protein was prepared underreducing conditions, on direct comparison to migration of a 36 kDaprotein) and an isoelectric point of 5.3±0.2 as determined based on itselution profile from a chromatofocusing column, and a molecular weightof 40 kDa (as determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis after the protein was prepared under reducingconditions, from a regression based on the log of molecular weight ofstandard proteins) and an isoelectric point of 3.9 as determined byisoelectric focusing electrophoresis from a regression of distanceversus the isoelectric point of standard proteins. For the otherfraction, a chitobiase portion elutes as a single peak at pH 4.3-4.6(variation between runs). The chitobiase portion is indicated bypolyacrylamide gel electrophoresis under non-reducing conditions tocontain an intensively staining protein which has chitobiase activityand has a molecular weight of 36 kDa (as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis after the protein wasprepared under reducing conditions, on direct comparison to migration ofa 36 kDa protein), and an isoelectric point of 4.4±0.2 as determinedbased on its elution profile from a chromatofocusing column, and amolecular weight of 40 kDa (as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis after the protein was prepared underreducing conditions, from a regression based on the log of the molecularweight of standard proteins), and an isoelectric point of 3.9 asdetermined by isoelectric focusing electrophoresis from a regression ofdistance versus isoelectric point of standard proteins. Chitobiase canbe separated from contaminating proteins by isoelectric focusing on aRotofor apparatus and consists of two proteins of similar isoelectricpoint. The larger of the two proteins (40 kDa as determined by sodiumdodecyl gel electrophoresis, after the protein was prepared underreducing conditions, from a regression based on the log of the molecularweight of standard proteins), stains more intensively following sodiumdodecyl sulfate-polyacrylamide gel electrophoresis and is denoted theprotein of the major band. The other chitobiase is denoted the proteinof the minor band. The protein of the major band can be obtained in pureform (i.e., uncontaminated by protein of the minor band), if thepurification proceeds over several weeks or if the chitobiase is driedin a Speedvac vacuum dryer, presumably since the protein of the minorband is degraded over time or during drying in the Speedvac.

As indicated above, a further embodiment of the invention hereininvolves a biologically pure (i.e., free of contaminating protein)composition containing endochitinase (enzyme that cleaves chitinrandomly) and chitobiase (enzyme that cleaves dimeric units from chitin)in a weight ratio ranging from 3:1 to 1:1.2, preferably ranging from 2:1to 1:1. This composition provides synergistic antifungal effect comparedto endochitinase or chitobiase alone and is considered to be a generaloccurrence, that is to be the case for all endochitinases and allchitobiases. Preferably the endochitinase is the endochitinase hereinand the chitobiase is the chitobiase herein.

The purified chitinases herein, and the biologically pure compositionsherein containing endochitinase and chitobiase, inhibitchitin-containing fungi and chitin-containing herbivorous insects. Thechitin-containing fungi inhibited by the purified chitinases hereininclude, for example, species from genera including Fusarium,Gliocladium, Rhizoctonia, Trichoderma, Uncinula, Ustilago, Erysiphe,Botrytis, Saccharomyces, Sclerotium, and Alternaria. Thechitin-containing herbivorous insects inhibited by the purifiedchitinases herein include, for example, Lepidoptera includingTrichoplusia ni (cabbage looper), Pieris rapae (imported cabbage worm),corn earworm, gypsy moth, pink boll worm, tobacco bollworm, diamondbackmoth, codling moth and spruce budworm; Coleoptera including Coloradopotato beetle, boll weevil, Mexican bean beetle and corn rootworm;Homoptera including citrus psylla, cotton aphid, peach-potato aphid andCalifornia red scale; Thysanoptera, including onion thrips; Orthopteraincluding migratory locusts; Hemiptera including rice stink bug; Dipteraincluding Hessian fly and cabbage root fly; Acari including European redmite, citrus red mite and two spotted mite; Siphonoptera includingLucerne flea; Isoptera including harvester termite; and Hymenopteraincluding leaf cutting ants.

Inhibition of the aforementioned by the purified chitinases herein orbiologically pure composition of endochitinase and chitobiase is readilycarried out by contacting the fungus or insect with the chitinase or thebiologically pure composition.

The purified chitinase or combinations thereof or biologically purecomposition as described above can be utilized as a solution in aconcentration, for example, of 50 ppm to 1000 ppm enzyme and applied inthe form of a spray, or as a solid wherein the chitinase ingredient(s)is (are) present together with an agriculturally acceptable adhesivecarrier, e.g., methyl cellulose or gum arabic, and applied as a powder.

Application can be, for example, to the seed, foliage or roots of aplant to be protected from a chitin-containing insect orplant-pathogenic fungus, or the soil surrounding said plant, or to achitin-containing fungus or insect to be inhibited.

The purified chitinases herein can also be used to isolate genes codingfor them. This can be carried out as follows:

Lyophillized mycelium obtained from Trichoderma harzianum P1 (ATCC74058) is ground into a fine powder and suspended in a lysis buffer. Thesuspension is treated with oligo dT cellulose resin which absorbs mRNA,which has a polyadenylated (poly A⁺) sequence at the 3' end. Unboundcellular debris, chromatin DNA, ribosomal RNA, membranes and othermolecules are removed by washing the resin. The mRNA is eluted with alow salt buffer and recovered by ethanol precipitation. This procedureyields 20-100 μg of poly A⁺ mRNA per gram of dried mycelium. An mRNAisolation kit is commercially available (e.g., from Invitrogen of SanDiego, Calif.) for this procedure.

Oligo dT primers are added to 10 μg poly A⁺ mRNA with the first cDNAstrand synthesized by adding reverse transcriptase and dNTP's. The mRNAis degraded from the mRNA:cDNA hybrid and the second cDNA strand issynthesized by adding dNTP's, DNA polymerase 1, T4 DNA polymerase and E.coli ligase. Linkers are added to the newly synthesized cDNA moleculesand the molecules are ligated into a viral expression vector, e.g.,λgtll, to form viral particles containing a cDNA library. Insertion ofthe linkered cDNA is upstream of the β-galactosidase stop codon. Thisformation of viral particles containing a cDNA library from the isolatedpoly A⁺ mRNA is readily carried out utilizing a commercially availablekit (e.g., from Invitrogen). The viral particles express the cDNA codingsequence when placed into E. coli.

E. coli, strain Y1090, is lytically infected with the viral particles.At the appropriate time, the plaque lawn plate is overlain with a coatedmembrane that stimulates expression of the β-galactosidase genecontaining the ligated cDNA molecule. Expressed fusion particles bind tothe membrane, which is probed with polyclonal antibodies specific forthe chitinase of interest. Detection of those plaques expressing thegenes of interest is determined using a colony screening system. Thoseplaques expressing the genes of interest are isolated.

The polyclonal antibodies used above are formed by injecting 25 μg ofpurified chitinase weekly into a rabbit, for a total of six injections.Total antibodies are isolated from the rabbit serum (Goding, J. W.,Monoclonal Antibodies, Academic Press, London, 1983), withcross-reactivity and specificity determined using Western blots(Burnett, W. N., Anal. Biochem., 112, 195-203 (1981).

The gene (cDNA insert) can be removed from the λgtll recombinant byusing the restriction enzyme, Not I.

The genes produced as above can be inserted into microorganisms by knownmethods, e.g. as in U.S. Pat. No. 4,751,081. The transgenicmicroorganisms can be used to produce chitinase or as biocontrol agents.

The genes produced as above can be also inserted by known methods intoplants (e.g., as described in European patent application 339,009) as adefense against chitinase-sensitive pests.

The Rotofor apparatus, mentioned above and used in the examples below,is manufactured by Bio Rad and is a free solution isoelectric focusingapparatus for preparative protein purification on the basis ofisoelectric point. It consists of a cylindrical horizontal focusingchamber which rotates about its axis to eliminate thermal andgravitational convection and can hold up to 58 ml of solution to beseparated. The chamber contains 19 polyester membrane screens arrangedin parallel within the focusing chamber. These screens allow theproteins to migrate during focusing but maintain separation patternsuntil the moment of harvesting. The solution containing the sample to beseparated is mixed with an appropriate ampholyte solution, and theadmixture is placed evenly in the horizontal apparatus. A current isapplied, and the ampholytes migrate to the position where they have anet zero electrical charge, and in this way a pH gradient is formed. Theproteins to be separated also migrate to the region containing the pHwhere they have a net zero charge (i.e., to the pH of their isoelectricpoint). Once this separation has occurred, separate samples arecollected from each of the 20 compartments provided by the 19 polyestermembrane screens by a vacuum sampling apparatus.

The invention is illustrated in the following specific examples:

EXAMPLE I

Modified Richard's medium (as described hereinbefore) containing 1%chitin from crab shells as the sole carbon source is placed in ten 250ml Erlenmeyer flasks (100 ml/flask) and sterilization is carried out byautoclaving. Two ml of a heavy suspension of conidia (approximately 10⁹/ml) of Trichoderma harzianum strain P1 (ATCC 74058) were used toinoculate each flask and the resulting cultures were grown on a rotaryshaker for 4 days at 25° C.

After four days of culturing, the hyphal mass was removed from themedium by centrifugation, and the supernatant (800 ml) was dialyzedagainst 50 mM KHPO₄ buffer pH 6.7 (MWCO 8,000) to remove small molecularweight molecules and then concentrated about 30-fold by applyingpolyethylene glycol (MW 35,000) to the outside of the dialysis tubing,to produce a concentrated broth.

The concentrated broth (approximately 30 ml), in two separate samples,was applied to a 5×60 cm Sephacryl S-300 column and eluted with 50 mMKHPO₄ buffer pH 6.7 containing 200 mM NaCl. Each elution fractionconsisted of about 8 ml. The elution profiles of enzyme activity areshown in FIG. 1. In FIG. 1, the X's denote the graph for % reduction inturbidity (endochitinase activity), the open boxes denote the graph forbiase activity and the filled-in diamonds denote the graph for nagaseactivity. As shown in FIG. 1, the void volume eluted at fraction 60, anda peak of nagase and biase activity eluted at about fraction 100. Atabout fraction 115 there was a peak of chitobiase activity andendochitinase activity, but little nagase activity. At fractions 160-180there was a broad peak containing only endochitinase activity. Thesefractions were combined into three sets, designated I, II and III, asshown on FIG. 1. Set I (approximately 120 ml) contained nagase andchitobiase activity. Set II (approximately 160 ml) contained chitobiaseactivity and endochitinase activity and a low level of nagase activity.Set III (approximately 200 ml) contained only endochitinase activity.Set II was combined with a similar fraction from another run on theSephacryl column. Set III was combined with a similar fraction fromanother run of the Sephacryl column. The Set II combined fractions weredialyzed against 25 mM imidazole-HCl buffer 6.7 and concentrated to 20to 60 ml. The Set III combined fractions were dialyzed against 25 mMimidazole-HCl buffer 6.7 and concentrated to 20 to 60 ml. Theseconcentrates were applied to 1.1×25 cm chromatofocusing columnsequilibrated with 25 mM imidazole buffer. The proteins were eluted withPolybuffer at a pH range of 6.7 to 4. The elution pattern for theconcentrate of combined Sets III is shown in FIG. 2 wherein the openboxes denote the graph for Fraction vs. pH and the filled-in diamondsdenote the graph for Fraction vs. % reduction in turbidity. Theendochitinase (turbidity reducing) activity from the concentrate ofcombined Sets III (fraction numbers 20 to 22 consisting of approximately24 ml) eluted at a single peak at pH 5.3±0.2. The purity of enzyme inthis fraction after dialysis and drying was confirmed by polyacrylamidegel electrophoresis under non-denaturing conditions; only a singleprotein band was detected with silver stain. The protein in this band isdetermined to have a molecular weight of 36 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis after protein was preparedunder reducing conditions, on direct comparison to migration of a 36 kDaprotein; a molecular weight of 40 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis after the protein is prepared underreducing conditions, from a regression based on the log of the molecularweight of standard proteins; an isoelectric point of 5.3±0.2 based onits elution profile from a chromatofocusing column; and an isoelectricpoint of 3.9 by isoelectric focusing electrophoresis from a regressionof distance versus isoelectric point of standard proteins. The elutionpattern for the concentrate of combined Sets II is shown in FIG. 3wherein the graph denoted by filled in diamonds is for nagase activity,the graph denoted by open boxes and a peak at about fraction 20 is forchitobiase activity, and the graph denoted by x's that progressesdownwardly is for Fraction vs. pH. As shown in FIG. 3, the fractionswith the greatest amount of chitobiase activity (fractions 19-22consisting approximately of 32 ml) eluted in a single peak at about pH4.6. In another run these fractions eluted in a single peak at about pH4.3. As shown in FIG. 3 these fractions contained some nagase activity.This peak (after dialysis and drying) was found by polyacrylamide gelelectrophoresis under non-reducing conditions to contain three proteinbands. Only one of these bands, the most intensively staining one, wasfound to have chitobiase activity. The protein of this band wasdetermined to have an isoelectric point of 4.4±0.2 (based on its elutionprofile from a chromatofocusing column), has an isoelectric point of 3.9by isoelectric focusing electrophoresis from a regression of distanceversus isoelectric point of standard proteins, was determined by sodiumdodecyl sulfate polyacrylamide gel electrophoresis after the protein wasprepared under reducing conditions to have a molecular weight of about36 kDa on direct comparison to migration of a 36 kDa protein, and has amolecular weight of 40 kDa by sodium dodecyl sulfate polyacrylamide gelelectrophoresis after the protein is prepared under reducing conditionsand the molecular weight determined from regression based on the log ofthe molecular weight of standard proteins. This protein is isolated fromthe other two present by gel filtration liquid chromatography on Bio-GelP-60 (Bio Rad Laboratories).

Comparative Example I

Chitinases isolated from pea, tomato and bean were reported to beendochitinases and to have molecular weights of 27-39 kDa andisoelectric points ranging from 8.87 to 9.4.

An enzyme from T. reesei which is an endochitinase was estimated to havea molecular weight of 58 kDa.

The enzyme system of Serratia marcescens strain QMB1466 (ATCC 990) wasreported to have chitobiase activity. This activity is associated, onsodium dodecyl sulfate polyacrylamide gel electrophoresis under reducingconditions, with two major protein bands of molecular weights at 52.5and 58 kDa and two minor protein bands of molecular weights of 21.5 and40.4 kDa. This strain is the one used in the working Example of U.S.Pat. No. 4,751,081 for chitinase activity.

EXAMPLE II

The purified endochitinase from Sets III in Example I (fraction numbers20 to 22 of FIG. 2) and purified chitobiase enzyme from Sets II inExample I (fraction numbers 19-22 in FIG. 3), that is the fractioncontaining the essentially pure endochitinase having a molecular weightof 36 kDa (as determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis after the protein was prepared under reducingconditions, on direct comparison to migration of a 36 kDa protein) andan isoelectric point of 5.3±0.2 based on its elution profile from achromatofocusing column (denoted endochitinase below) and the fractioncontaining the protein having chitobiase activity, a molecular weight of36 kDa (as determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis after the protein was prepared under reducingconditions, on direct comparison to migration of a 36 kDa protein) andan isoelectric point of 4.4±0.2 as determined based on its elutionprofile from a chromatofocusing column (denoted biase below), wereassayed for antifungal activity against Fusarium sp. and Trichodermaharzianum (ATCC 20847). This was carried out on the dialyzed, driedfractions, as follows: A solution containing 500 ppm of enzyme(dialyzed, dried fraction) in distilled water was prepared and wassterilized by filtration through a 0.45 μm filter. From each sterilizedcomposition an assay mixture was prepared that contained 100 μl of thesterilized composition, 100 μl of 3× potato dextrose broth (DifcoLaboratories), and 100 μl of a fungal spore suspension (10⁶ /ml). Eachassay mixture was incubated. Results of spore germination after a 12hour incubation were as follows:

                  TABLE 1                                                         ______________________________________                                        Enzyme       Organism  Spore Germination %                                    ______________________________________                                        None (control)                                                                             Fusarium  85                                                       Biase Fusarium 63                                                             Endochitinase Fusarium 58                                                     None (control) Trichoderma 49                                                 Biase Trichoderma 20                                                          Endochitinase Trichoderma 22                                                ______________________________________                                    

These data show that spore germination was inhibited by the enzymesolutions.

Other effects were noted as follows: Germ tube growth was inhibitedafter 24 hours of incubation of the Trichoderma strain by both the biasesolution and the endochitinase solution. Among spores producing germtubes, the average length (of 15 measured) for germ tubes germinating inthe absence of the enzyme was 198 μm, while the average length of germtubes in the presence of the biase was 55 μm and the average length ofgerm tubes in the presence of the endochitinase was 58 μm.

EXAMPLE III

Filter sterilized broth prepared from Trichoderma harzianum strain P1(ATCC 74058) was compared to filter-sterilized broth from T. virde 105,T. koningii 8, T. koningii 417, T. koningii VS023, T. harzianum 1295-22,and Gliocladium virens VS031 as follows: In each case, a diet was madeconsisting of the following in parts by weight: 57 parts Trichoderma orGliocladium broth, 11.4 parts wheat germ, 2 parts casein, 1.4 partsagar, 1 part Vitamin Premix (Hoffman-LaRoche #26862), 0.8 parts WessonSalt Mixture (Bio Serv, Inc.), 0.2 parts sorbic acid, 0.1 parts methylparaben, and 26 parts distilled water. Larvae of T. ni and P. rapae wereprovided with the diet, ad libitum from neonate until the controlsreached the ultimate instar, and then all larvae were weighed. Only twoof the fungal strains, G. virens VS031 and T. harzianum P1 (ATCC 74058),reduced the growth of both the larval T. ni and P. rapae below 60%. Thefilter-sterilized broth from each of these two strains was dialyzed(MWCO 8,000) to remove small molecular weight molecules and incorporatedinto the artificial diet as described above. Again, the broth from thesetwo strains significantly reduced the growth of larval T. ni and P.rapae. The ammonium sulfate precipitable protein in thefilter-sterilized, dialyzed broth from both strains was tested forbiological activity against larval T. ni and P. rapae as above. Thechitinase activity of the original P1 broth was not precipitated whilethat of the VS031 broth was. The ammonium sulfate precipitated proteinfrom strain VS031 significantly reduced larval growth while that ofstrain P1 did not.

EXAMPLE IV

A medium containing 10 g KNO₃, 5 g KH₂ PO₄, 2.5 g MgSO₄ ×7H₂ O, 2 mgFeCl₃, 10 g crab shell chitin (Sigma Chemical Co.), 150 ml of vegetablejuice cocktail (V8 juice), 10 g polyvinylpyrollidone (Polyclar AT, GAFCorp.) and 1000 ml water was adjusted to pH 6 and then sterilized byautoclaving.

The medium was placed in Erlenmeyer flasks (100 ml per 250 ml flask) andeach flask was inoculated with a spore suspension of Trichodermaharzianum strain P1 (ATCC 74058) to give about 5×10⁶ conidia finalconcentration and the inoculated flasks were placed on a rotary shakerat 150 RPM at 25° C. for 4 or 5 days. The liquid was separated from thebiomass by centrifugation at 8000×g for 10 minutes and residualparticulates were removed through a glass fiber filter to provide aculture filtrate containing the enzymes of interest.

The culture filtrate was dialyzed against 50 mM KPO₄ buffer pH 6.7 (6liters of buffer per liter of culture filtrate) overnight at 4° C. withstirring. After dialysis, the dialysis tubes were placed in polyethyleneglycol (35,000 MW, Fluka Chemika-Biochemika) until the volume wasreduced 15- to 25-fold.

The concentrated culture filtrate (approximately 15 ml) in two separatesamples was applied to a 5×60 cm chromatography column packed withSephacryl S-300 (Pharmacia LKB Biotechnology) equilibrated with 50 mMKPO₄ buffer pH 6.7 containing 200 mM NaCl and 0.02% NaN₃ and eluted withthe same buffer. The concentrated culture filtrate and elution bufferwere applied from the bottom of the column using a pump set to deliver2.3 ml per minute, and fractions were collected every 5 minutes.

The elution profiles of enzyme activity are shown in FIG. 4. In FIG. 4,the x's denote endochitinase activity, the open boxes denote chitobiaseactivity and the filled in diamonds denote nagase activity. As shown inFIG. 4, at fractions 92-100 there was a peak of chitobiase activitytogether with endochitinase and nagase activity; these fractions weredesignated set II. As shown in FIG. 4, fractions 120-150 contained mostof the endochitinase activity; these fractions were designated Set III.

The fractions of Set III (345 ml) were pooled, and the combinedfractions were concentrated to 25 ml in dialysis tubing immersed inpolyethylene glycol (molecular weight of 35,000) overnight againstapproximately a 10-fold volume of 25 mM imidazole-HCl buffer pH 7. Theconcentrate was applied to a 1×30 cm chromatofocusing column packed withPBE 94 (Pharmacia LKB Biotechnology) equilibrated with 25 mM imidazolebuffer. The proteins were eluted with Polybuffer (Pharmacia LKBBiotechnology), at a pH range of 7 to 4. The elution pattern forconcentrated fractions of set III is shown in FIG. 5. In FIG. 5, theopen boxes denote endochitinase activity and the x's denote pH.Electrophoresis on native, sodium dodecyl sulfate and isoelectricfocusing gels showed a single protein. Activity, as determined byfluorescence of methylumbelliferyl substrate, corresponded to theprotein band on isoelectric focusing and native gels and showed theprotein to be an endochitinase. The molecular weight of the protein wasdetermined to be 40 kDA (as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis after the protein was prepared underreducing conditions, from a regression based on the log of the molecularweight of standard proteins) and the isoelectric point of the proteinwas determined to be 3.9 by isoelectric focusing electrophoresis from aregression of distance versus isoelectric point of standard proteins.The specific activity of the purified endochitinase was determined to be0.86 units/μg protein with the turbidity reducing assay and 2.2 nkat/μgprotein when nitrophenyl-β-D-N,N',N"-triacetylchitotriose was used asthe substrate. The specific activity of the endochitinase in theoriginal culture filtrate could not be determined because said filtratecontains an inhibitor of endochitinase activity. The endochitinase wasdetermined to have optimum activity at about pH 4 with a gradual declineto about pH7.

The fractions of Set II (92 ml) were pooled, and the combined fractionswere concentrated in dialysis tubing immersed in polyethylene glycol(molecular weight of 35000) to approximately 25 ml and then dialyzedovernight against approximately a 10-fold volume of 25 mM imidazole-HClbuffer pH7. The concentrate was applied to a 1×30 cm chromatofocusingcolumn packed with PBE94 (Pharmacia LKB Biotechnology) equibrated with25 mM imidazole buffer. The proteins were eluted with Polybuffer(Pharmacia LKB Biotechnology), at a pH range of 7 to 4. The elutionpattern for the concentrated fractions of set II is shown in FIG. 6. InFIG. 6, the filled in diamonds denote chitobiase activity, the openboxes denote nagase activity and the x's denote pH. In addition tochitobiase and nagase activity, a small overlapping peak of glucanaseactivity was detected by hydrolysis of nitrophenyl-β-D-glucopyranoside.Since the glucanase activity eluted in fractions after 28, fractions 25to 27 were pooled for further purification. The pooled fractions werefurther purified by separation according to isoelectric point on aRotofor apparatus (Bio-Rad Laboratories) employing Bio-Lyte 3/5ampholyte at 10% v/v of the total volume and collecting peak fractionsand purifying these on the Rotofor apparatus. The separation pattern forsamples collected from the Rotofor apparatus is shown in FIG. 7. In FIG.7 the filled-in diamonds denote chitobiase activity, the open boxesdenote nagase activity and the x's denote pH. Fractions 2 to 8 werecollected and contained chitobiase activity and no endochitinase ornagase activity. The chitobiase was determined to consist of two closelyspaced protein bands on sodium dodecyl sulfate, isoelectric focusing andnative gels. The larger of the two bands provided a more intensivelystaining protein band and was the major product. These bands correspondto activity bands as determined by overlay of fluorescentenzyme-specific substrate on isoelectric focusing and native gels. Thechitobiase of the major band was determined to have a molecular weightof 40 kDa (as determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis after the protein was prepared under reducingconditions, from regression based on the log of the molecular weight ofstandard proteins) and an isoelectric point of 3.9 as determined byisoelectric focusing electrophoresis from a regression of distanceversus isoelectric point of standard proteins. The chitobiase of theminor band was determined to have a molecular weight of 36 kDa (asdetermined by sodium dodecyl sulfate polyacrylamide gel electrophoresisafter the protein was prepared under reducing conditions, fromregression based on the log of the molecular weight of standardproteins) and an isoelectric point of 3.9 as determined by isoelectricfocusing electrophoresis from a regression of distance versusisoelectric point of standard proteins. The chitobiase of the minor bandwas present at a level of no more than 40% by weight (total chitobiasebasis).

In runs where the purification step was carried out over a period ofseveral weeks or where the purified chitobiase sample was dried in aSpeedvac vacuum drying apparatus, the minor band was not present andessentially pure chitobiase of the major band was obtained.

Specific activities of the chitobiase were determined at various stagesof processing with the following results.

                  TABLE 2                                                         ______________________________________                                                          Specific activity                                                                         Recovery                                          Purification step (nkat/mg protein) (%)                                     ______________________________________                                        Culture filtrate  0.54        100                                               After dialysis and 12.3 39                                                    concentration                                                                 Set II after Sephacryl 26.2 29                                                chromatography                                                                After chromatofocusing 51.7 13                                                After isoelectric focusing 127 12                                             in the Rotofor (major band                                                    protein)                                                                    ______________________________________                                    

The above results show that the activity of the chitobiase of the majorband is purified more than 75 times after chromatofocusing and more than200 times after isoelectric focusing in the Rotofor.

The chitobiase of the major band was determined to have an optimumactivity between pH 4 and pH 7.

EXAMPLE V

The essentially pure endochitinase of Example IV and essentially purechitobiase of the major band of Example IV and a 1:1 (w/w) mixturethereof were assayed for antifungal activity against Botrytis cinerea,Fusarium solani, Ustilago avenae, Uncinula necator, Trichodermaharzianum (ATCC 20847), Saccharomyces cerevisiae, Fusarium graminearum,Trichoderma harzianum strain P1 (ATCC 74058) and Pythium ultimum strainP4. This was carried out as follows: Equal volumes (100 μl) of fungussuspension (10⁶ spores or cells/ml), medium and enzyme test solution ofvarious amounts of dialyzed dried fraction in distilled water sterilizedby filtration, were mixed. The medium in all cases except for Ustilagoavenae was 3×potato dextrose broth (Difco Laboratories). In the case ofUstilago avenae the medium was a 3× concentration of Koller and Wubbenmedium, i.e., a medium containing 50 mM 2-(N-morpholino)ethanesulfonicacid containing (g/l) dipotassium hydrogen phosphate, 1.0; magnesiumsulfate, 0.5; ammonium nitrate, 1.0; potassium chloride, 0.5; ferrousammonium sulfate, 0.1; glucose, 10.0; yeast extract 1.0; and 0.5 ml of amicronutrient solution (which contained (mg/liter) sodium tetraboratedecahydrate, 1500; manganese sulfate monohydrate, 15; zinc sulfateheptahydrate, 2500; cuprous sulfate pentahydrate, 100; sodium molybdate,1000) wherein the medium was adjusted to pH 6.1 and sterilized byfiltration. The assay mixtures were incubated for 22-48 hours at 20-25°C.

The results are given in the following Table 3 where "ge/re" stands forspore germination or sporidia or cell replication and "elong" stands forgerm tube elongation and ED₅₀ means the dose for 50% inhibition of sporegermination or sporidia or cell replication in the case of ge/re and 50%reduction in the length of germ tubes produced from surviving conidia ascompared to length of control germ tubes (produced without enzymespresent) and "n.d." stands for not determined and "chitob+endoc" standsfor the 1:1 mixture of chitobiase and endochitinase.

                                      TABLE 3                                     __________________________________________________________________________                 ED.sub.50 for enzymes (μg × ml.sup.-1)                               chitobiase                                                                             endochitinase                                                                         chitob + endoc                                  FUNGI        ge/re                                                                              elong                                                                             ge/re                                                                             elong                                                                             ge/re                                                                             elong                                       __________________________________________________________________________    Botrytis cinerea                                                                           152  125 41  58  10  24                                            Fusarium solani 165 168 110 67 30 28                                          Ustilago avenae 179 --  135 --  34 --                                         Uncinula necator 180 173 35 30 13 10                                          Trichoderma harzianum 62 162 90 35 n.d. n.d.                                  Saccharomyces cerevisiae 490 --  535 --  400 --                               Fusarium graminearum 125 132 100 70 n.d. n.d.                                 Trichoderma harzianum (P1) >1000 >1000 >1000 >1000 >1000 >1000                Pythium ultimum >1000 >1000 >1000 >1000 >1000 >1000                         __________________________________________________________________________

EXAMPLE VI

The synergy between the essentially pure endochitinase of Example IV andthe essentially pure chitobiase of the major band of Example IV isdemonstrated according to the formula in Richter, D. L., Pestic. Sci.19:309-315, 1987. In accordance with that formula, if synergism existsE_(O) (xA+yB)>E_(O) (x+y)A, and >E_(O) (x+y)B, where E_(O) stands forpercentage inhibition and x is the concentration of A (endochitinaseherein) in the enzyme mixture and y is the concentration of B(chitobiase herein) in the enzyme mixture. In this Example, assays werecarried out as in Example V except that 50 μg/ml of endochitinase wasused in one case and 50 μg/ml of chitobiase was used in another case and50 μg of a 1:1 mixture of endochitinase was used in the third case andpercent inhibition was determined. Since the total enzyme concentrationwas the same in the three cases, synergism exists according to theformula in Richter if the percent inhibition for the mixture is greaterthan that for either of the individual cases.

The data obtained is set forth in Table 4 below.

                  TABLE 4                                                         ______________________________________                                                enzyme(s) (total concentration = 50 μg/ml)                                                         endochitinase +                                  endochitinase chitobiase chitobiase                                           (E.sub.O = (E.sub.O = (E.sub.O =                                             Fungus % inhibition) % inhibition) % inhibition)                            ______________________________________                                        Botrytis cinerea                                                                        60          20        97                                              Fusarium solani 20 12.5 70                                                    Uncinula necator 72 12 93                                                     Ustilago avenae 7.5 5.5 55                                                  ______________________________________                                    

As indicated by the data, not only is synergism present according to theformula of Richter since the percent inhibition of the mixture isgreater than the percent inhibition for the same total concentration ofeither of the components of the mixture but also the percent inhibitionfor the mixture exceeds that of the sum of the percent inhibitions forthe same total concentrations of the individual components.

EXAMPLE VII

Various combinations of the essentially pure endochitinase of Example IVand the essentially pure chitobiase of the major band of Example IV(weight ratios of 12:3, 9:6, 7.5:7.5, 6:9 and 3:12) were assayed forantifungal activity against Botrytis cinerea. Equal volumes (100 μl) ofspore suspension (10⁶ /ml), 3× potato dextrose broth medium and enzymetest solution (15 ppm of enzyme combination), in distilled water,sterilized by filtration, were mixed. The assay mixtures were incubatedat 25° C. for 4 hours. The results are set forth in FIG. 8 where thegraph delineated by filled in blocks relates to % inhibition of sporegermination and the graph delineated by open blocks relates to percentinhibition of hyphal elongation. In FIG. 8 the data at 15:0 and 0:15 isrepresented by bars where the solid bars represent percent inhibition ofspore germination and the open bars represent percent inhibition ofhyphal elongation.

EXAMPLE VIII

The gene for the endochitinase herein was isolated as follows:

I. Isolation of Messenger Ribonucleic Acid (mRNA)

Biomass of induced Trichoderma harzianum strain P1 was asepticallyharvested in a sterile Buchner funnel lined with Miracloth (Calbiochem,La Jolla, Calif.) and washed with RNase-free water. The biomass wasplaced in a sterile disposable petri plate then quick-frozen by placingthe plate in liquid nitrogen. The frozen biomass was dried by placing itin a prechilled lyophilizer and establishing a vacuum. The shelftemperature of the lyophilizer was maintained at -5° C. while thecondenser temperature was set at -50° C.

Approximately 1 gram of lyophilized mycelium was resuspended in 20 ml ofa guanidinium solution (5 M guanidinium thiocyanate; 50 mM Tris-HCl, pH7.5; 10 mM Na₂ EDTA; 5% B-mercaptoethanol, added after theaforementioned solution had been sterilely filtered and just before use)and incubated at room temperature for 10 minutes. The supernatant wasremoved by centrifuging at 12,000×g for 10 minutes at 12° C.N-lauroylsarcosine was added to a final concentration of 2% and themixture was incubated at 65° C. for 2 minutes. The aqueous phase wasextracted with an equal volume of phenol/chloroform/isoamyl alcohol(25:24:1), then with an equal volume of chloroform/isoamyl alcohol(24:1). Sodium acetate was added to a final concentration of 300 mM andthe RNA precipitated by the addition of 2.5 volumes of cold ethanol.mRNA was purified by direct affinity absorption to oligo dT celluloseresin using the FastTrack mRNA Isolation Kit (Invitrogen; San Diego,Calif.). This procedure yielded about 15 μg of poly A⁺ mRNA.

II. Insertion of cloned 2'-Deoxyribonucleic Acid (cDNA) into aBacteriophage Expression Vector, λgtII

About 5 μg of poly A⁺ RNA was used for first strand cDNA synthesis usingan oligo dT primer and reverse transcriptase. The mRNA-cDNA hybrid wasconverted into double-stranded cDNA by the addition of RNase H, DNApolymerase I and E. coli DNA ligase. The newly formed double-strandedcDNA molecules was blunt-ended with T4 DNA polymerase and EcoR I (Not I)adaptors were added using T4 DNA ligase. Overhang ends of the adaptorswere phosphorylated with T4 polynucleotide kinase. The adapted cDNAswere selected by electroelution from agarose to provide moleculesranging in size from 900 base-pairs (bp) to 7,000 bp, then ligated intolambda vector arms that had been EcoR I cut and dephosphorylated. Theviral vector was encapsulated in a protein coat with approximately 60%of the vector containing a foreign insert in a total of 1.3×10⁵ viralparticles.

III. Production of Polyclonal Antibodies Specific for Endochitinase fromT. harzianum, Strain P1

Two, eight week old female Flemish giant/Chinchilla rabbits weresubcutaneously injected each week, with about 25 μg of pureendochitinase per injection, for a total of six weeks. Totalimmunoglobulins (Ig), containing polyclonals specific for theendochitinase, were recovered from rabbit serum by precipitationfollowing addition of ammonium sulfate (50% of saturation) of the rabbitserum. Specificity for endochitinase was determined using ELISA andWestern blotting. The two screening techniques demonstrated that theantibody that was specific for pure endochitinase was IgG. An E. colilysate, at a 1:10 dilution, was added to the IgG solution and themixture was incubated overnight at 4° C. and diluted 1:400 before beingused to screen the cDNA library of T. harzianum strain P1.

IV. Screening the cDNA Library

A culture of E. coli, strain Y1090, was mixed with the phage particleswhich resulted in lytic infection, poured into a plate and incubateduntil viral plaques developed. The plate was overlain with anitrocellulose membrane, that had been soaked in a solution ofisopropyl-β-D-thiogalactopyranoside (IPTG) and allowed to air dry. Themembrane position on the plate was marked, then carefully removed andblocked with 1% bovine serum albumin in TBS (20 mM Tris-HCl, pH 7.4: 150mM NaCl). The membrane was probed with the polyclonal antibody solution,then washed (3×) in TBS containing 0.05% Tween-20 (TBST). Plaquesexpressing fusion proteins with affinity for the polyclonal antibodieswere detected by hybridization to a goat antirabbit IgG (1:5000 dilutionin TBS of the monovalent immunoglobulin that has a conjugated alkalinephosphatase enzyme). The membrane was washed (3×) in TBST, thendeveloped in an alkaline phosphatase buffer (100 mM Tris, pH 9.5, 100 mMNaCl, 50 mM MgCl₂ containing 225 μg 4-nitro blue tetrazolium chlorideand 150 μg 5-bromo-4-chloro-3-indolyl-phosphate per ml of buffer). Thoseviral plaques expressing the protein of interest were further purified,then stored.

V. Assaying for Enzymatically Active Fusion Protein

Enzyme assays were performed with fusion proteins, produced by thepurified plaques to see if they were active. Lysogens were formed in E.coli, Y1089, with active fusion protein observed in polyacrylamide gelsoverlain with 4-methylumbelliferyl β-D-N,N",N"-triacetylchitotriose.

VI. Isolation of Bacteriophage Particles

Bacteriophage particles with affinity for the polyclonal antibody wereisolated from lytically infected E. coli strain Y1090. Lyticallyinfected bacterial cells were plated on LB agar and after plaquesdeveloped, about 6 ml of SM medium (50 mM Tris-HCl pH 7.5, 100 mM NaCl,10 mM MgSO₄, 0.01% gelatin) was added to the plate, which was placed ona rotary shaker at 4° C. After an overnight incubation, the liquid wasremoved and chloroform (7% final concentration) was mixed into theliquid, with the upper aqueous phase removed by centrifugation. DNaseand RNase (final concentration of 1 μg/ml of each enzyme) were added andthe mixture shaken at 37° C. for 1 hr. Chloroform (7%) was again added,the mixture was centrifuged, and the aqueous phase containing the phageparticles recovered. The phage particles were ready for storage in 1%chloroform or for isolation of DNA. The phage particles constitute λgtllrecombinant containing a cDNA fragment of the gene encoding for theendochitinase herein and a sample in TE buffer (100 mM Tris-HCl, pH7.6,+1 mM EDTA) was deposited with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852, on Jul. 6, 1992, under theterms of the Budapest Treaty and has been assigned accession number ATCC55338.

VII. Isolation of Viral DNA

NaCl was added to suspension of bacteriophage particles to give a 1 Msolution and incubated 1 hr in an ice bath. The mixture was centrifugedat 11,000×g for 10 min at 4° C. to remove cellular debris, and thesupernatant recovered. Polyethylene glycol (MW 8000) was added to give afinal concentration of 10% (w/v), and the mixture placed in an ice bathfor 1 hr. The bacteriophage particles were recovered by centrifugationat 11,000×g for 10 min at 4° C., and the supernatant removed anddiscarded. The bacteriophage particles were resuspended in SM medium, anequal volume of chloroform was added, mixed, centrifuged and the aqueousphase retained. The bacteriophage particles were recovered bycentrifugation at 25,000×g for 2 hr at 4° C. SM medium (1 to 2 ml) wasadded and the mixture was incubated at 4° C. with shaking. Proteinase K(50 μg/ml) and sodium dodecyl sulfate (final concentration 0.5% from a20% stock solution) were added and mixed by inverting the mixtureseveral times in a closed tube. The mixture was incubated at 50° C. for1 hr, cooled to room temperature and an equal volume ofbuffer-equilibrated phenol was added. The tube was inverted severaltimes to mix, the phases were separated by centrifugation and the upperphase recovered by gentle suction with a wide-bore pipet. The aqueousphase was extracted with an equal volume of phenol/chloroform/isoamylalcohol (25:24:1), then with an equal volume of chloroform/isoamylalcohol (24:1). Sodium acetate was added to a final concentration of 300mM and the DNA precipitated by the addition of 2.5 volumes of coldethanol.

VIII. Removal of the cDNA Insert

The foreign DNA was recovered from the DNA purified from λgtll bydigestion with Not I. only adaptors on either side of the cDNA were cut,with nucleotide sequence comprising fragment of the gene coding forendochitinase released in an undigested state. The released segment wasestimated to be 1150 base pairs in length, based on estimations of sizeversus that of standards following electrophoretic separations.

IX. Obtaining the Sequence of the Gene

When the released segment was sequenced, it was found to be 1095 basepairs long, including 8 base pairs at the 5' end that derive from λgtlland 13 base pairs in the poly A tail. It also contained 209 base pairsat the 3' end which did not code for amino acids, so the releasedsegment was not of sufficient length to code for the entire amino acidsequence of a 40 kDa protein. Moreover, the segment contained no ATGstart codon, or other indication of a start to the gene. Therefore, itwas concluded that the segment was missing a portion at the 5' end ofthe gene. The sequence of the 1074 base pairs of the segmentcorresponding to portion at the 3' end of the gene, i.e., of fragment atthe 3' end of the gene excluding the 13 base pairs of the poly A tail,is set forth in the Sequence Listing as SEQ ID NO:3. Sequencing wascarried out by the Sanger (dideoxy) method.

A 20 base pair primer consisting of base pairs 70 through 89 downstreamfrom the 5' end of the fragment having the sequence set forth in theSequence Listing as SEQ ID NO:3, is set forth in the Sequence Listing asSEQ ID NO:4. This 20 base pair primer is designated hereinafter as thenested primer. The nested primer was used in combination with forward orreverse λgtll primers. Small aliquots from the cDNA library (10⁵ phage)were amplified by lytic infection of E. coli Y1090 and subjected topolymerase chain reaction in λgtll that contained both the nested primerand either the forward or reverse λgtll primers. Any amplified segmentcoding for the remainder of the gene would contain at its 3' end thefirst 89 base pairs of the 5' end of the original gene fragment, plusthe remainder of the gene. Moreover, there is a Pst I restriction site15 base pairs from the 5' end of the original gene fragment so digestionof the proper amplified segment with Pst I would provide a 74 base pairsegment.

Following amplification, several segments were obtained, including a 628base pair fragment that appeared with both nested and forward or nestedand reverse primers. This is expected since the intact gene likely wouldhave inserted in both orientations into λgtll. When digested with Pst I,this fragment gave the expected 74 base pair segment. Therefore, the 628base pair portion was ligated into pCRII (TA cloning kit, Invitrogen)and sequenced by the Sanger (dideoxy) method. The sequence is set forthin the sequence listing as SEQ ID NO:5. The first 59 base pairs of this628 base pair fragment arose from λgtll since the reverse primer was 59base pairs from the insertion point in the vector. The 89 base pairoverlap region was identical between the original 1074 base pairfragment and the 628 base pair product. Thus, the sequence of the entiregene is indicated from the isolated fragments. The sequence of theentire gene is 1554 base pairs long excluding the 13 base pairs in thepoly A tail and the λgtll sequence at the 5' end of the 628 base pairfragment and this 1554 base pair sequence is set forth in the SequenceListing as SEQ ID NO:1. The overlap region referred to is at positions481-569. The entire sequence includes a 73 base pair untranslated leadersequence, an open reading frame encoding a putative protein of 424 aminoacids and 209 base pair untranslated 3' region. The sequence of theprotein is set forth as a feature in SEQ ID NO:1 and is also set forthin SEQ ID NO:2. The 424 amino acid protein includes a 35 amino acidleader sequence that must be cleaved to give rise to a mature protein of389 amino acids. The 35 amino acid leader sequence has manycharacteristics of a signal peptide including a core of hydrophobicamino acids. The amino acid sequence deduced for the mature proteinprovides a mass of 42.66 kDa and has an isoelectric point of 4.53, whichare reasonably close to the values of 40 kDa and 3.9 estimated from gelelectrophoresis of the isolated protein; the slight variations mayresult from secondary modifications of the protein, e.g., low levels ofcarbohydrates attached to the protein.

X. Isolating the Gene

A 20 base pair primer consisting of base pairs 1520 through 1539 of thegene was prepared. It has the sequence set forth in the Sequence Listingas SEQ ID NO:6. This primer was used together with a reverse λgtllprimer to screen aliquots of the cDNA library in λgtll, and an aliquotthat gave a 1550 base pair band upon electrophoresis (on gels, bandsdiffering in fewer than about 50 base pairs cannot be distinguished innucleotides of this size). Performance of a Southern blot on the gel andprobing with an 81 base pair fragment labeled with digoxigenin(Boehringer Mannheim, Mannheim, Germany) made up of bases 489-569verified that this band was homologous with the probe and that theentire gene is contained in λgtll clone. The appropriate clone ispurified by excising the plaque giving the positive reaction, using thisto reinfect E. coli, again isolating and probing plaques and continuingthis until all plaques are homologous with the probe. The presence ofthe entire gene is verified by detection of the entire sequence viaamplification with polymerase chain reaction using forward and reverseλgtll primers.

EXAMPLE IX

Purified Endochitinase from G. virens Strain 41 (ATCC 20906)

Synthetic medium is made up containing 680 mg KH₂ PO₄, 870 mg K₂ HPO₄,200 mg KCl, 1 g NH₄ NO₃, 200 mg CaCl₂, 200 mg MgSO₄.7H₂ O, 2 mg FeSO₄, 2mg ZnSO₄, 2 mg MnSO₄, 42 g moist purified colloidal chitin (prepared asdescribed in Vessey, J. C., et al, Trans. Br. Mycol. Soc. 60:710-713,1973), 5 g sucrose, in 1 L distilled water, final pH 6.0.

100 ml of the synthetic medium is placed in a 250 ml Erlenmeyer flask.

The flask is inoculated with conidia grown by inoculation of potatodextrose agar (conidia of Gliocladium virens ATCC 20906) to provide 10⁷conidia ml⁻¹ medium and the admixture is incubated at 25° C. for 5 or 7days on a rotary shaker at 200 rpm. The culture filtrate is harvested bycentrifugation at 8000×g for 10 minutes and removal of residualparticulates by filtration through a glass fiber filter.

The purified endochitinase is isolated from the culture filtrate asdescribed below with all steps being carried out at 4° C. except forconcentration which was carried out at room temperature.

The filtered culture filtrate is transferred into dialysis tubing (6,000to 8,000 Da cutoff) and dialyzed overnight against 50 mM potassiumphosphate buffer pH 6.7 (5 L buffer L⁻¹ culture filtrate), and thenconcentrated 30-40 fold by placing the tubing in solid polyethyleneglycol (35,000 MW; Fluka Chemika-Biochemica, Buchs, Switzerland). Theconcentrate is then applied to a gel filtration column (5×60 cm)containing Sephacryl S-300 HR (Pharmacia LKB Biotechnology, Uppsala,Sweden) equilibrated with 50 mM potassium phosphate buffer pH 6.7containing 200 mM NaCl. The material from 1 L of culture medium ischromatographed separately in two samples on Sephacryl S-300 HR.Fractions, approximately 8 ml each, are eluted with 1500 ml of 50 mMpotassium phosphate buffer containing 200 mM NaCl. A first peak betweenfractions 70 and 120 contains high levels of chitobiosidase andN-acetyl-β-glucosaminidase activity. A second peak with endochitinase,β-1,3-glucanase, and chitobiosidase activity is detected in fractions120 to 140. Fractions 140 to 160 contain endochitinase activity;proteins in this region are apparently not separated on the basis ofmolecular weight, but adsorbed to the gel matrix since they elute at orgreater than the total column volume. The fractions 140 to 160 from thefirst sample and similar fractions from the other, showing onlyendochitinase activity, are pooled. The pooled fractions (160 ml) aretransferred into dialysis tubing (6,000 to 8,000 Da cut-off) andconcentrated 30- to 40-fold by placing the tubing in solid polyethyleneglycol (35,000 MW; Fluka Chemika-Biochemika, Buchs, Switzerland) anddialyzed overnight against a 20-fold volume of 25 mM ethanolamine-HClbuffer pH 8.7. The sample (about 25 ml) is then applied to achromatofocusing column (1×30 cm) packed with PBE94 (Pharmacia LKB), andequilibrated with the same buffer used for dialysis. The column iseluted at a flow rate of 50 ml h⁻¹ with Polybuffer 96 (Pharmacia LKB)diluted 1:10 and adjusted to pH 7.0 with HCl according to themanufacturer's direction. A sharp peak at pH 8.0 containingendochitinase activity is detected in the eluted fractions. The peakfractions are pooled and the pooled fractions (about 40 ml) are dialyzedfirst against a 20-fold volume of 1M NaCl and then against a 40-foldvolume of distilled water to remove Polybuffer, and concentrated to avolume of 2 ml in a collodion bag system (10,000 Daj UH 100/1,Schleicher & Schuell Inc., Keene, N.H.). The sample (2 ml), i.e., theconcentrated fractions, are applied to compartments 15 and 16 (pH8.0-8.5) of a Rotofor isoelectric focusing cell (Bio-Rad, Richmond,Calif.) loaded with 35 ml distilled water containing 2% of BiolyteAmpholytes pH 3-10 (Bio-Rad), run at 12 W constant power, at atemperature of 40° C., after one hour of prefocusing run, and the run iscontinued for 5 hours. The fractions (each about 2 ml) are collected andassayed for endochitinase activity. The peak fractions containhomogeneous endochitinase as shown by the presence of a single proteinband upon SDS-PAGE and upon Native PAGE. A single fluorescent activityband is observed following overlay of the native gel with themethylumbelliferyl substrate. This activity corresponds to the positionof the single protein band detected with coomassie blue and silverstain.

The peak (active) fractions are pooled, dialyzed against 1M NaCl andthen against distilled water as described above, and concentrated todryness in a Speedvac apparatus. The enzyme is stored at -20° C. andreconstituted in an appropriate volume of sterilized distilled water foruse.

The endochitinase from G. virens strain 41 (ATCC 20906) has a molecularweight of 41 kDa as determined by sodium dodecyl sulfate polyacrylamidegel electrophoresis after the protein was prepared under reducingconditions from a regression based on the log of the molecular weight ofstandard proteins, and an isoelectric point of 7.8 as determined byisoelectric focusing electrophoresis from a regression of distanceversus isoelectric point of standard proteins, as described in U.S.patent application Ser. No. 08/184,115.

EXAMPLE X Antifungal Use

In this example, the endochitinase is the above-described endochitinasefrom Gliocladium virens strain 41 having accession No. 20906.

Assays were carried out for inhibition of the plant pathogen Botrytiscinerea as described in Lorito, M., et al, Phytopathology, Vol. 83, No.3, 303-307 (1993) except that the spores were harvested and suspended in5 mM sodium acetate buffer to improve spore germination, the assay wasperformed in flat bottomed wells in microtiter plates and observed after24 hr. with an inverted microscope and 50 μg/ml of the antibioticampicillum was used to prevent bacterial growth during the assay.

Results of % inhibition of hyphal elongation are given in Table 5 below:

                  TABLE 5                                                         ______________________________________                                        Enzyme(s) and Concentration(s)                                                                   % Inhibition                                               ______________________________________                                        Endochitinase at 1 μg/ml                                                                      5.5                                                          Endochitinase at 5 μg/ml 24                                                Endochitinase at 10 μg/ml 54                                             ______________________________________                                    

Results of % inhibition of spore germination are given in Table 6 below:

                  TABLE 6                                                         ______________________________________                                        Enzyme(s) and Concentration(s)                                                                   % Inhibition                                               ______________________________________                                        Endochitinase at 1 μg/ml                                                                      5.5                                                          Endochitinase at 5 μg/ml 25                                                Endochitinase at 10 μg/ml 51                                             ______________________________________                                    

As indicated above, the term "chitobiase" is used herein to mean enzymethat cleaves dimeric unit from chitin. Others use this term to refer toan enzyme that cleaves chitobiose (the dimer) to monomeric units. Sothat there can be no confusion in the minds of those reading the termherein without having referred to the definition herein, the term"chitin 1,4-β-chitobiosidase" or the term "chitobiosidase" can be usedin place of the term "chitobiase" herein to mean enzyme that cleavesdimeric unit from chitin.

SEQ ID NO:7 constitutes base pairs 443-487 of SEQ ID NO:1. SEQ ID NO:8constitutes base pairs 566-595 of SEQ ID NO:1. SEQ ID NO:9 constitutesamino acids 123-138 of SEQ ID NO:2. SEQ ID NO:10 constitutes amino acids165-174 of SEQ ID NO:2.

Variations in the invention will be obvious to those skilled in the art.Therefore, the invention is defined by the claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 10                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1554 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 74..1345                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GCTCTTTTCA GCAGCAACTT CTTCCTTTCA AAGCATCTCT TGACAACCTT TG -            #CTGAATCT     60                                                                 - - CAAACACTTC ACC ATG TTG GGC TTC CTC GGA AAA TC - #C GTG GCC CTG CTT           109                                                                                     Met Leu - #Gly Phe Leu Gly Lys Ser Val Ala Leu Leu                              1 - #              5    - #              10                   - - GCT GCG CTG CAG GCC ACT CTC ATT TCT GCA TC - #T CCT GTA ACT GCA AAC          157                                                                       Ala Ala Leu Gln Ala Thr Leu Ile Ser Ala Se - #r Pro Val Thr Ala Asn                    15         - #         20         - #         25                      - - GAC GTC TCT GTT GAG AAG AGA GCC AGT GGA TA - #C GCA AAC GCC GTC TAC          205                                                                       Asp Val Ser Val Glu Lys Arg Ala Ser Gly Ty - #r Ala Asn Ala Val Tyr                30             - #     35             - #     40                          - - TTC ACC AAC TGG GGT ATT TAC GGC CGC AAC TT - #C CAG CCT CAG AAC CTG          253                                                                       Phe Thr Asn Trp Gly Ile Tyr Gly Arg Asn Ph - #e Gln Pro Gln Asn Leu            45                 - # 50                 - # 55                 - # 60       - - GTC GCG TCG GAC ATC ACT CAT GTC ATC TAC TC - #G TTC ATG AAC TTC CAA          301                                                                       Val Ala Ser Asp Ile Thr His Val Ile Tyr Se - #r Phe Met Asn Phe Gln                            65 - #                 70 - #                 75              - - GCA GAC GGC ACT GTC GTC TCT GGA GAT GCC TA - #C GCC GAT TAT CAG AAG          349                                                                       Ala Asp Gly Thr Val Val Ser Gly Asp Ala Ty - #r Ala Asp Tyr Gln Lys                        80     - #             85     - #             90                  - - CAC TAT GAC GAC GAT TCT TGG AAC GAC GTC GG - #T AAC AAT GCG TAC GGC          397                                                                       His Tyr Asp Asp Asp Ser Trp Asn Asp Val Gl - #y Asn Asn Ala Tyr Gly                    95         - #        100         - #        105                      - - TGT GTG AAG CAG CTG TTC AAG CTG AAG AAG GC - #C AAC CGC AAC TTG AAG          445                                                                       Cys Val Lys Gln Leu Phe Lys Leu Lys Lys Al - #a Asn Arg Asn Leu Lys               110              - #   115              - #   120                          - - GTT ATG CTT TCC ATC GGT GGC TGG ACC TGG TC - #C ACC AAC TTT CCT TCT          493                                                                       Val Met Leu Ser Ile Gly Gly Trp Thr Trp Se - #r Thr Asn Phe Pro Ser           125                 1 - #30                 1 - #35                 1 -      #40                                                                              - - GCA GCA AGC ACC GAT GCC AAC CGC AAG AAC TT - #T GCC AAG ACT GCC        ATC      541                                                                    Ala Ala Ser Thr Asp Ala Asn Arg Lys Asn Ph - #e Ala Lys Thr Ala Ile                          145  - #               150  - #               155              - - ACC TTC ATG AAG GAC TGG GGT TTC GAT GGT AT - #T GAC GTC GAT TGG GAG          589                                                                       Thr Phe Met Lys Asp Trp Gly Phe Asp Gly Il - #e Asp Val Asp Trp Glu                       160      - #           165      - #           170                  - - TAC CCC GCC GAT GAT ACC CAG GCC ACC AAC AT - #G GTT CTT CTG CTC AAG          637                                                                       Tyr Pro Ala Asp Asp Thr Gln Ala Thr Asn Me - #t Val Leu Leu Leu Lys                   175          - #       180          - #       185                      - - GAG ATC CGA TCT CAG CTA GAT GCC TAT GCT GC - #G CAA TAC GCT CCG GGC          685                                                                       Glu Ile Arg Ser Gln Leu Asp Ala Tyr Ala Al - #a Gln Tyr Ala Pro Gly               190              - #   195              - #   200                          - - TAC CAC TTC CTT CTT TCC ATT GCT GCC CCC GC - #T GGC CCA GAG CAC TAC          733                                                                       Tyr His Phe Leu Leu Ser Ile Ala Ala Pro Al - #a Gly Pro Glu His Tyr           205                 2 - #10                 2 - #15                 2 -      #20                                                                              - - TCT TTC CTG CAC ATG TCC GAC CTT GGC CAA GT - #T CTC GAC TAT GTC        AAC      781                                                                    Ser Phe Leu His Met Ser Asp Leu Gly Gln Va - #l Leu Asp Tyr Val Asn                          225  - #               230  - #               235              - - CTC ATG GCC TAC GAC TAT GCT GGT TCT TGG AG - #C AGC TAC TCC GGA CAC          829                                                                       Leu Met Ala Tyr Asp Tyr Ala Gly Ser Trp Se - #r Ser Tyr Ser Gly His                       240      - #           245      - #           250                  - - GAT GCC AAC TTG TTT GCC AAC CCG TCC AAC CC - #C AAC TCT TCA CCA TAC          877                                                                       Asp Ala Asn Leu Phe Ala Asn Pro Ser Asn Pr - #o Asn Ser Ser Pro Tyr                   255          - #       260          - #       265                      - - AAC ACC GAT CAA GCT ATC AAG GAC TAT ATC AA - #G GGA GGT GTT CCC GCA          925                                                                       Asn Thr Asp Gln Ala Ile Lys Asp Tyr Ile Ly - #s Gly Gly Val Pro Ala               270              - #   275              - #   280                          - - AGC AAG ATC GTT CTT GGC ATG CCC ATC TAC GG - #A CGA GCT TTT GAG AGC          973                                                                       Ser Lys Ile Val Leu Gly Met Pro Ile Tyr Gl - #y Arg Ala Phe Glu Ser           285                 2 - #90                 2 - #95                 3 -      #00                                                                              - - ACC GGT GGC ATT GGC CAG ACC TAC AGT GGA AT - #T GGA TCT GGA AGC        TGG     1021                                                                    Thr Gly Gly Ile Gly Gln Thr Tyr Ser Gly Il - #e Gly Ser Gly Ser Trp                          305  - #               310  - #               315              - - GAG AAC GGT ATT TGG GAC TAC AAG GTT CTT CC - #C AAG GCC GGC GCC ACA         1069                                                                       Glu Asn Gly Ile Trp Asp Tyr Lys Val Leu Pr - #o Lys Ala Gly Ala Thr                       320      - #           325      - #           330                  - - GTC CAG TAT GAC TCT GTC GCA CAG GCA TAC TA - #C AGC TAT GAC CCC AGC         1117                                                                       Val Gln Tyr Asp Ser Val Ala Gln Ala Tyr Ty - #r Ser Tyr Asp Pro Ser                   335          - #       340          - #       345                      - - AGC AAG GAG CTC ATC TCT TTC GAT ACC CCT GA - #C ATG ATC AAC ACC AAG         1165                                                                       Ser Lys Glu Leu Ile Ser Phe Asp Thr Pro As - #p Met Ile Asn Thr Lys               350              - #   355              - #   360                          - - GTC TCT TAC CTC AAG AAC CTC GGC CTG GGA GG - #C AGC ATG TTC TGG GAA         1213                                                                       Val Ser Tyr Leu Lys Asn Leu Gly Leu Gly Gl - #y Ser Met Phe Trp Glu           365                 3 - #70                 3 - #75                 3 -      #80                                                                              - - GCT TCT GCT GAC AAG ACT GGC TCT GAC TCC TT - #G ATC GGA ACA AGC        CAC     1261                                                                    Ala Ser Ala Asp Lys Thr Gly Ser Asp Ser Le - #u Ile Gly Thr Ser His                          385  - #               390  - #               395              - - AGA GCT TTG GGA AGC CTA GAC TCC ACT CAG AA - #C TTG CTG AGC TAC CCC         1309                                                                       Arg Ala Leu Gly Ser Leu Asp Ser Thr Gln As - #n Leu Leu Ser Tyr Pro                       400      - #           405      - #           410                  - - AAC TCC CAG TAT GAT AAC ATC CGA AGC GGT CT - #C AAC TAGAGATCTT              1355                                                                       Asn Ser Gln Tyr Asp Asn Ile Arg Ser Gly Le - #u Asn                                   415          - #       420                                             - - TCTTCTTCTT ATCTTTTTCT TTTACTTCCC CTATGGTTGT ACCAACATTT CA -             #CACACGTT   1415                                                                 - - ATGCGAAACG ATTATGCAGG GAGCGTTATT TTTTAGTAAA TAGTTGCCCT TT -            #GAGATATA   1475                                                                 - - TGAACCTGTA CATAAAGAAC TACTAGCAGT ATATAAGGAG ACATGCAGGA TC -            #TCTAGAAT   1535                                                                 - - TGACTTCCAT GCTTTCCTC             - #                  - #                     155 - #4                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 424 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Leu Gly Phe Leu Gly Lys Ser Val Ala Le - #u Leu Ala Ala Leu Gln        1               5 - #                 10 - #                 15              - - Ala Thr Leu Ile Ser Ala Ser Pro Val Thr Al - #a Asn Asp Val Ser Val                   20     - #             25     - #             30                  - - Glu Lys Arg Ala Ser Gly Tyr Ala Asn Ala Va - #l Tyr Phe Thr Asn Trp               35         - #         40         - #         45                      - - Gly Ile Tyr Gly Arg Asn Phe Gln Pro Gln As - #n Leu Val Ala Ser Asp           50             - #     55             - #     60                          - - Ile Thr His Val Ile Tyr Ser Phe Met Asn Ph - #e Gln Ala Asp Gly Thr       65                 - # 70                 - # 75                 - # 80       - - Val Val Ser Gly Asp Ala Tyr Ala Asp Tyr Gl - #n Lys His Tyr Asp Asp                       85 - #                 90 - #                 95              - - Asp Ser Trp Asn Asp Val Gly Asn Asn Ala Ty - #r Gly Cys Val Lys Gln                  100      - #           105      - #           110                  - - Leu Phe Lys Leu Lys Lys Ala Asn Arg Asn Le - #u Lys Val Met Leu Ser              115          - #       120          - #       125                      - - Ile Gly Gly Trp Thr Trp Ser Thr Asn Phe Pr - #o Ser Ala Ala Ser Thr          130              - #   135              - #   140                          - - Asp Ala Asn Arg Lys Asn Phe Ala Lys Thr Al - #a Ile Thr Phe Met Lys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Trp Gly Phe Asp Gly Ile Asp Val Asp Tr - #p Glu Tyr Pro Ala        Asp                                                                                             165  - #               170  - #               175             - - Asp Thr Gln Ala Thr Asn Met Val Leu Leu Le - #u Lys Glu Ile Arg Ser                  180      - #           185      - #           190                  - - Gln Leu Asp Ala Tyr Ala Ala Gln Tyr Ala Pr - #o Gly Tyr His Phe Leu              195          - #       200          - #       205                      - - Leu Ser Ile Ala Ala Pro Ala Gly Pro Glu Hi - #s Tyr Ser Phe Leu His          210              - #   215              - #   220                          - - Met Ser Asp Leu Gly Gln Val Leu Asp Tyr Va - #l Asn Leu Met Ala Tyr      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp Tyr Ala Gly Ser Trp Ser Ser Tyr Ser Gl - #y His Asp Ala Asn        Leu                                                                                             245  - #               250  - #               255             - - Phe Ala Asn Pro Ser Asn Pro Asn Ser Ser Pr - #o Tyr Asn Thr Asp Gln                  260      - #           265      - #           270                  - - Ala Ile Lys Asp Tyr Ile Lys Gly Gly Val Pr - #o Ala Ser Lys Ile Val              275          - #       280          - #       285                      - - Leu Gly Met Pro Ile Tyr Gly Arg Ala Phe Gl - #u Ser Thr Gly Gly Ile          290              - #   295              - #   300                          - - Gly Gln Thr Tyr Ser Gly Ile Gly Ser Gly Se - #r Trp Glu Asn Gly Ile      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Trp Asp Tyr Lys Val Leu Pro Lys Ala Gly Al - #a Thr Val Gln Tyr        Asp                                                                                             325  - #               330  - #               335             - - Ser Val Ala Gln Ala Tyr Tyr Ser Tyr Asp Pr - #o Ser Ser Lys Glu Leu                  340      - #           345      - #           350                  - - Ile Ser Phe Asp Thr Pro Asp Met Ile Asn Th - #r Lys Val Ser Tyr Leu              355          - #       360          - #       365                      - - Lys Asn Leu Gly Leu Gly Gly Ser Met Phe Tr - #p Glu Ala Ser Ala Asp          370              - #   375              - #   380                          - - Lys Thr Gly Ser Asp Ser Leu Ile Gly Thr Se - #r His Arg Ala Leu Gly      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ser Leu Asp Ser Thr Gln Asn Leu Leu Ser Ty - #r Pro Asn Ser Gln        Tyr                                                                                             405  - #               410  - #               415             - - Asp Asn Ile Arg Ser Gly Leu Asn                                                      420                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1074 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - CAACTTTCCT TCTGCAGCAA GCACCGATGC CAACCGCAAG AACTTTGCCA AG -             #ACTGCCAT     60                                                                 - - CACCTTCATG AAGGACTGGG GTTTCGATGG TATTGACGTC GATTGGGAGT AC -            #CCCGCCGA    120                                                                 - - TGATACCCAG GCCACCAACA TGGTTCTTCT GCTCAAGGAG ATCCGATCTC AG -            #CTAGATGC    180                                                                 - - CTATGCTGCG CAATACGCTC CGGGCTACCA CTTCCTTCTT TCCATTGCTG CC -            #CCCGCTGG    240                                                                 - - CCCAGAGCAC TACTCTTTCC TGCACATGTC CGACCTTGGC CAAGTTCTCG AC -            #TATGTCAA    300                                                                 - - CCTCATGGCC TACGACTATG CTGGTTCTTG GAGCAGCTAC TCCGGACACG AT -            #GCCAACTT    360                                                                 - - GTTTGCCAAC CCGTCCAACC CCAACTCTTC ACCATACAAC ACCGATCAAG CT -            #ATCAAGGA    420                                                                 - - CTATATCAAG GGAGGTGTTC CCGCAAGCAA GATCGTTCTT GGCATGCCCA TC -            #TACGGACG    480                                                                 - - AGCTTTTGAG AGCACCGGTG GCATTGGCCA GACCTACAGT GGAATTGGAT CT -            #GGAAGCTG    540                                                                 - - GGAGAACGGT ATTTGGGACT ACAAGGTTCT TCCCAAGGCC GGCGCCACAG TC -            #CAGTATGA    600                                                                 - - CTCTGTCGCA CAGGCATACT ACAGCTATGA CCCCAGCAGC AAGGAGCTCA TC -            #TCTTTCGA    660                                                                 - - TACCCCTGAC ATGATCAACA CCAAGGTCTC TTACCTCAAG AACCTCGGCC TG -            #GGAGGCAG    720                                                                 - - CATGTTCTGG GAAGCTTCTG CTGACAAGAC TGGCTCTGAC TCCTTGATCG GA -            #ACAAGCCA    780                                                                 - - CAGAGCTTTG GGAAGCCTAG ACTCCACTCA GAACTTGCTG AGCTACCCCA AC -            #TCCCAGTA    840                                                                 - - TGATAACATC CGAAGCGGTC TCAACTAGAG ATCTTTCTTC TTCTTATCTT TT -            #TCTTTTAC    900                                                                 - - TTCCCCTATG GTTGTACCAA CATTTCACAC ACGTTATGCG AAACGATTAT GC -            #AGGGAGCG    960                                                                 - - TTATTTTTTA GTAAATAGTT GCCCTTTGAG ATATATGAAC CTGTACATAA AG -            #AACTACTA   1020                                                                 - - GCAGTATATA AGGAGACATG CAGGATCTCT AGAATTGACT TCCATGCTTT CC - #TC             1074                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - GTAGCTTTGG GGTCAGGAAG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 628 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - TTGACACCAG ACCAACTGGT AATGGTAGCG ACCGGCGCTC AGCTGGAATT CG -             #CGGCCGCG     60                                                                 - - CTCTTTTCAG CAGCAACTTC TTCCTTTCAA AGCATCTCTT GACAACCTTT GC -            #TGAATCTC    120                                                                 - - AAACACTTCA CCATGTTGGG CTTCCTCGGA AAATCCGTGG CCCTGCTTGC TG -            #CGCTGCAG    180                                                                 - - GCCACTCTCA TTTCTGCATC TCCTGTAACT GCAAACGACG TCTCTGTTGA GA -            #AGAGAGCC    240                                                                 - - AGTGGATACG CAAACGCCGT CTACTTCACC AACTGGGGTA TTTACGGCCG CA -            #ACTTCCAG    300                                                                 - - CCTCAGAACC TGGTCGCGTC GGACATCACT CATGTCATCT ACTCGTTCAT GA -            #ACTTGGAA    360                                                                 - - GCATACGGCA CTGTCGTCTC TGGAGATGCC TACGCCGATT ATCAGAAGCA CT -            #ATGACGAC    420                                                                 - - GATTCTTGGA ACGACGTCGG TAACAATGCG TACGGCTGTG TGAAGCAGCT GT -            #TCAAGCTG    480                                                                 - - AAGAAGGCCA ACGGCAACTT GAAGGTTATG CTTTCCATCG GTGGCTGGAC CT -            #GGTCCACC    540                                                                 - - AACTTTCCTT CTGCAGCAAG CACCGATGCC AACCGCAAGA ACTTTGCCAA GA -            #CTGCCATC    600                                                                 - - ACCTTCATGA AGGACTGGGG TTTCGATG         - #                  - #                628                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GCAGGATCTC TAGAATTGAC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - AAGGTTATGC TTTCCATCGG TGGCTGGACC TGGTCCACCA ACTTT   - #                      - #45                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GATGGTATTG ACGTCGATTG GGAGTACCCC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Leu Lys Val Met Leu Ser Ile Gly Gly Trp Th - #r Trp Ser Thr Asn Phe      1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Asp Gly Ile Asp Val Asp Trp Glu Tyr Pro                                  1               5   - #                10                                    __________________________________________________________________________

What is claimed is:
 1. An isolated DNA molecule encoding a fungalendochitinase comprising an amino acid sequence of SEQ. ID. No. 9 orSEQ. ID. No.
 10. 2. An isolated DNA molecule according to claim 1,wherein the endochitinase comprises an amino acid sequence of SEQ. ID.No.
 9. 3. An isolated DNA molecule according to claim 1, wherein theendochitinase comprises an amino acid sequence of SEQ. ID. No.
 10. 4. Avector containing the DNA molecule according to claim
 2. 5. A vectorcontaining the DNA molecule according to claim
 3. 6. A host cellcontaining the DNA molecule according to claim
 2. 7. A host cellcontaining the DNA molecule according to claim
 3. 8. A host cellaccording to claim 6, wherein said host cell is a microorganism.
 9. Ahost cell according to claim 7, wherein said host cell is amicroorganism.
 10. A host cell according to claim 6, wherein said hostcell is a plant cell.
 11. A host cell according to claim 7, wherein saidhost cell is a plant cell.
 12. A transgenic plant containing the DNAmolecule according to claim
 2. 13. A transgenic plant containing the DNAmolecule according to claim
 3. 14. An isolated DNA molecule encoding anendochitinase and comprising the nucleotide sequence of SEQ. ID. No. 7or the nucleotide sequence of SEQ. ID. No.
 8. 15. An isolated DNAmolecule according to claim 14 comprising the nucleotide sequence ofSEQ. ID. No.
 7. 16. An isolated DNA molecule according to claim 14comprising the nucleotide sequence of SEQ. ID. No.
 8. 17. A vectorcontaining the DNA molecule according to claim
 14. 18. A vectorcontaining the DNA molecule according to claim
 15. 19. A vectorcontaining the DNA molecule according to claim
 16. 20. A host cellcontaining the DNA molecule according to claim
 14. 21. A host cellcontaining the DNA molecule according to claim
 15. 22. A host cellcontaining the DNA molecule according to claim
 16. 23. A host cellaccording to claim 20, wherein said host cell is a microorganism.
 24. Ahost cell according to claim 20, wherein said host cell is a plant cell.25. A transgenic plant containing the DNA molecule according to claim14.
 26. A transgenic plant containing the DNA molecule according toclaim
 15. 27. A transgenic plant containing the DNA molecule accordingto claim
 16. 28. An isolated DNA molecule encoding a fungalendochitinase having an amino acid sequence comprising SEQ.ID. No. 2.29. A vector containing the DNA molecule according to claim
 28. 30. Ahost cell containing the DNA molecule according to claim
 28. 31. A hostcell according to claim 30, wherein said host cell is a microorganism.32. A host cell according to claim 30, wherein said host cell is a plantcell.
 33. A transgenic plant containing the DNA molecule according toclaim 28.